Map of pRSET A, B, and C - Thermo Fisher Scientific
Contents
1. 10 x 10 ml 15544 034 Miller s LB Broth Base Luria Broth Base 500 e 12795 027 powder imMedia Amp Liquid 20 pouches Q600 20 200 ml medium imMedia Amp Agar 20 pouches Q601 20 8 10 plates UltraPure Sodium Dodecyl Sulfate SDS 500 e 15525 017 UltraPure DNase RNase Free Water 500 ml 10977 015 Overview Introduction Regulation of Expression of the Gene of Interest Regulation of Expression of T7 RNA Polymerase Introduction The pRSET vectors are pUC derived expression vectors designed for high level protein expression and purification from cloned genes in E coli High levels of expression of DNA sequences cloned into the pRSET vectors are made possible by the presence of the T7 promoter In addition DNA inserts are positioned downstream and in frame with a sequence that encodes an N terminal fusion peptide This sequence includes an ATG translation initiation codon a polyhistidine tag that functions as a metal binding domain in the translated protein a transcript stabilizing sequence from gene 10 of phage T7 the Xpress epitope and the enterokinase cleavage recognition sequence The metal binding domain of the fusion peptide allows simple purification of recombinant proteins by Immobilized Metal Affinity Chromatography with Invitrogen s ProBond resin available in bulk see page v The enterokinase cleavage recognition site in the fusion peptide located between the metal binding2. Add 1 mM IPTG to induce expression Grow at 37 C with shaking until the optimal time point determined by the pilot expression is reached Harvest the cells by centrifugation 3000 x g for 10 minutes at 4 C 7 Atthis point you may proceed directly to purification or store the cells for future use at 80 C 11 Recipes SOB For 1 Liter SOC For 1 Liter LB For 1 Liter Appendix To 950 ml of deionized water add 20 0 g Tryptone 5 0 g Yeast Extract 0 5 g NaCl 186 0 mg KCl 1 Mix the solution until dissolved 2 Adjust the pH to 7 0 with 5 N NaOH approximately 0 2 ml 3 If making solid media for plates or top agar add 15 g of agar after adjusting the pH 4 Adjust the volume to 1000 ml and sterilize by autoclaving 5 Once autoclaved add 10 ml of sterile 1 M Mg e g 10 ml of sterile 1 M MgCl or sterile 1 M MgSO Follow recipe as per SOB After autoclaving let cool to about 60 C and add 10 ml of 50 glucose Mix the media well Component liquid plates top agar Tryptone 10g 10g 10g Yeast Extract 58g 5g 5g NaCl 10g 10g 10g Agar 15g 7g 1 Combine the tryptone yeast extract and NaCl with 950 ml of deionized water Mix the solution until dissolved 2 Adjust the pH to 7 0 with 5 N NaOH will take about 0 2 ml If making solid media for plates or top agar add the appropriate amount of agar after adjusting the pH 3 Adjust volume to 1 liter with water Sterilize by auto
3. GGT ATG GCT AGC ATG ACT Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr T7 gene 10 leader Xpress Epitope BamH Xho Sac 1 1 IW GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT dcG AGC TCG Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys gAsp Pro Ser Ser EK recognition site K cleavage site Bgl Il Pst Pvull KpnlNcol EcoRI BstB Hind Ill N hd I l AGA TCT GCA GCT GGT ACC ATG GAA TTC GAA GCT TGA TCCGGCTGCT AACAAAGCCC Arg Ser Ala Ala Gly Thr Met Glu Phe Glu Ala T7 reverse priming site GAAAGGAAGC TGAGTTGGCT GCTGCCACCG CTGAGCAATA ACTAGCATAA Continued on next page Cloning into pRSET A B and C continued Multiple Cloning Site of pRSET C T7 promoter Below is the multiple cloning site for pRSET C Restriction sites are labeled to indicate the actual cleavage site The boxed nucleotides indicate the variable region Sequencing and functional testing have confirmed the multiple cloning site The complete sequence of pRSET C is available for downloading at www invitrogen com or from Technical Support see page 18 For a map and description of the features of pRSET C please refer to pages 14 15 RBS 21 AATACGACTC ACTATAGGGA GACCACAACG GTTTCCCTCT AGAAATAATT TTGTTTAACT TTAAGAAGGA Polyhistidine 6xHis region l 91 GATATACAT ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT Met Arg Gly Ser His His His His His
4. T7 gene 10 leader Xpress Epitope BamH Es GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT CGA TGG GGA Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp Arg Trp Gly EK recognition site EK cleavage site Abol Sac Bg Il Pst Pvull KpnlNcol EcoRI BstB Hind Ill Neil NI TCC GAG CTC GAG ATC TGC AGC TGG TAC l CAT GGA ATT CGA AGC TTG ATC CGG CTG CTA Ser Glu Leu Glu Ile Cys Ser Trp Tyr His Gly ile Arg Ser Leu Ile Arg Leu Leu T7 reverse priming site l ACA AAG CCC GAA AGG AAG CTG AGT TGG CTG CTG CCA CCG CTG AGC AAT AAC TAG CAT Thr Lys Pro Glu Arg Lys Leu Ser Trp Leu Leu Pro Pro Leu Ser Asn Asn His Continued on next page Cloning into pRSET A B and C continued Multiple Cloning Below is the multiple cloning site for pRSET B Restriction sites are labeled to Site of pRSET B indicate the actual cleavage site The boxed nucleotides indicate the variable 21 91 148 205 261 region Sequencing and functional testing have confirmed the multiple cloning site The complete sequence of pRSET B is available for downloading at www invitrogen com or from Technical Support see page 18 For a map and description of the features of pRSET B please refer to pages 14 15 T7 promoter RBS AATACGACTC ACTATAGGGA GACCACAACG GTTTCCCTCT AGAAATAATT TTGTTTAACT TTAAGAAGGA Polyhistidine 6xHis region GATATACAT ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT
5. cells by centrifuging at 4000 rpm for 10 minutes in a 4 C rotor Sorvall GSA 4 Resuspend the pellet in 10 ml of ice cold 50 mM CaCl Keep the cells on ice for at least 30 minutes 5 Centrifuge the CaCl treated cells in a 4 C rotor Sorvall SS 34 at 4000 rpm for 5 minutes Gently resuspend the cells in 4 ml of ice cold 50 mM CaCh Keep the cells on ice 6 Aliquot 100 ul of the CaCl treated cells for each transformation into a prechilled microcentrifuge tube Store the cells at 80 C for long term storage 7 For transformation take one tube of 100 ul of competent cells prepared above and add the plasmid DNA 10 100 ng to the cells Incubate the cells on ice for 30 minutes 8 Heat shock cells at 42 C for 45 seconds in a water bath Return the tube s to ice for 2 minutes 9 Add 1 ml of SOC media and incubate the culture s for 45 minutes at 37 C with vigorous shaking gt 200 cycles minute in a rotary shaker 10 Plate the appropriate amount of cells onto SOB plates containing the appropriate antibiotic selection for the plasmid for pRSET vectors use ampicillin Note When selecting for transformants in BL21 DE3 pLysS include 35 pg ml chloramphenicol in the plate For your convenience One Shot TOP10F or BL21 DE3 pLysS competent cells are available for high efficiency transformation See page v for more information 17 Technical Support Web Resources Visit the Invitrogen website at www invitrogen
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7. next page Overview continued Regulation of T7 RNA Polymerase by T7 Lysozyme Experimental Outline There is always some basal level expression of T7 RNA polymerase If a toxic gene is cloned downstream of the T7 promoter basal expression of this gene may lead to reduced growth rates cell death or plasmid instability T7 lysozyme produced from pLysS or pLysE has been shown to bind to T7 polymerase and inhibit transcription This activity is exploited to reduce basal levels of T7 RNA polymerase T7 lysozyme is a bifunctional enzyme In addition to its T7 RNA polymerase binding activity it also cleaves a specific bond in the peptidoglycan layer of the E coli cell wall This activity increases the ease of cell lysis by freeze thaw cycles prior to purification The table below describes the basic steps needed to clone and express your protein using pRSET A B and C For more details please refer to the page s indicated Step Action Page 1 Propagate and maintain the empty pRSET A B and C vectors by 3 transforming them into a recA endA E coli host i e TOP10F 2 Develop a cloning strategy to ligate your gene of interest into 4 7 pRSET A B or C 3 Ligate your gene of interest into pRSET transform into TOP10F 6 and select on 50 100 pg ml ampicillin 4 Sequence your construct to ensure that it is in frame with the 7 N terminal peptide Perform a pilot expression using IPTG for induct
8. supernatant to a fresh labeled tube To 100 ul of supernatant sample add an equal volume of 2X SDS PAGE sample buffer Resuspend the pellet in 100 pl of 1X SDS PAGE sample buffer Load 10 20 ul of each of the supernatant and pellet samples after boiling for 5 minutes on an appropriate SDS PAGE gel and electrophorese Stain the gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the recombinant protein Use the uninduced culture as a negative control From this expression experiment determine the optimal time after IPTG induction to harvest the cells In addition you may perform a western blot to confirm that the overexpressed band is your desired protein see next page Use the positive control to confirm that growth and induction were performed properly The pRSET lacZ vector should produce an 120 kDa protein when induced with IPTG Expression of your protein with the N terminal tag will increase the size of your protein by approximately 3 kDa Be sure to account for any additional amino acids between the tag and your protein Continued on next page Expression continued Detecting Recombinant Fusion Proteins Expressing Recombinant Protein Troubleshooting To detect expression of your recombinant fusion protein by western blot analysis you may use antibodies against the appropriate epitope available from Invitrogen see page v for ordering information or an anti
9. Grow the E coli strain overnight in SOB medium overnight with antibiotic selection when appropriate Combine 0 85 ml of the overnight culture with 0 15 ml of sterile glycerol Vortex and transfer to a labeled cryovial Freeze the tube in liquid nitrogen or dry ice ethanol bath and store at 80 C Accessory Products Introduction The tables below lists related products that may be used with pRSET A B and C Product Application Quantity Cat No One Shot TOP10F cells Chemically competent cells for 20 x 50 pl C3030 03 transformation One Shot Chemically competent cells for 20 x 50 pl C6060 03 BL21 DE3 pLysS cells transformation One Shot Chemically competent cells for 20 x 50 ul C6565 03 BL21 DE3 pLysE cells transformation One Shot BL21 DE3 cells Chemically competent cells for 20 x 50 ul C6000 03 transformation Anti Xpress Antibody Detection of recombinant proteins 50 ul R910 25 Anti Xpress HRP Detection of recombinant proteins 50 pl R911 25 Antibody Anti HisG Antibody Detection of recombinant proteins 50 pl R940 25 Anti HisG HRP Antibody Detection of recombinant proteins 50 pl R941 25 Anti HisG AP Antibody Detection of recombinant proteins 125 ul R942 25 ProBond Resin Purification of recombinant proteins 50 ml R801 01 150 ml R801 50 EnterokinaseMax Removal of N terminal peptide 250 units E180 01 EK Away Removal of EnterokinaseMax 7 5 ml R180 01 ProBond Pur
10. His Gly Met Ala Ser Met Thr T7 gene 10 leader Xpress H Epitope BamH I 148 GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC GAC GAT GAC GAT AAG GAT GGA TGG ATC Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys gASp Arg Trp Ile Xho EK recognition site K cleavage site Bgl Il Pst Pvull Kpnl Ncol EcoRI BstB Hind Ill Z l Ll NI l l 205 CGA CCT CGA GAT CTG CAG CTG GTA CCA TGG AAT TCG AAG CTT GAT CCG GCT GCT AAC Arg Pro Arg Asp Leu Gln Leu Val Pro Trp Asn Ser Lys Leu Asp Pro Ala Ala Asn T7 reverse priming site 262 AAA GCC CGA AAG GAA GCT GAG TTG GCT GCT GCC ACC GCT GAG CAA TAA CTA GCA Lys Ala Arg Lys Glu Ala Glu Leu Ala Ala ALa Thr Ala Gln Gln Ligation Transformation Once you have determined a cloning strategy digest the appropriate version of pRSET with the selected restriction enzyme Ligate your gene of interest into pRSETA B or C using standard molecular biology techniques After ligating your gene of interest into the appropriate pRSET vector transform the ligation mixture into competent TOP10F A detailed protocol for making competent TOP10F cells and using them for transformation is provided in the Appendix on page 17 Select 10 20 clones and analyze for the presence and orientation of your insert Continued on next page Cloning into pRSET A B and C continued N gio 7 Ke K d Nous gt Making Frozen Glycerol Stocks We recommend
11. Z 5911 nucleotides T7 promoter bases 20 39 6xHis tag bases 112 129 T7 gene 10 leader bases 133 162 Xpress epitope bases 169 192 lacZ ORF bases 199 3258 T7 reverse priming site bases 295 314 T7 transcription terminator bases 3270 3399 f1 origin bases 3470 3925 bla promoter bases 3957 4061 Ampicillin bla resistance gene ORF bases 4056 4916 pUC origin bases 3930 5866 C Transformation Protocol for TOP10F and BL21 DE3 pLysS Introduction Protocol This protocol is provided for your convenience Other protocols may be suitable Use the table below to select the appropriate medium for use with TOP10F or BL21 DE3 pLysS Strain Maintenance Medium pRSET Selection Medium TOP10F LB 10 pg ml tetracycline LB 50 pg ml ampicillin BL21 DE3 pLysS LB 35 pg ml LB 50 pg ml ampicillin chloramphenicol 35 ug ml chloramphenicol 1 Take the desired stab and streak out a small portion on the appropriate maintenance medium and incubate at 37 C overnight The stab should remain viable for several months when stored at 4 C in the dark We recommend making a frozen glycerol stock for long term storage see page 7 2 Pick a single colony and transfer it into 100 ml of SOB medium in a 1 liter flask see page 12 for media recipes Incubate the flask at 37 C with vigorous shaking gt 200 cycles minute in a rotary shaker 3 When the OD q reaches approximately 0 5 collect the
12. bable Cause Possible Solution No or low Insert ligated into wrong Check sequence carefully and determine expression reading frame which vector pRSET A B or C is appropriate with the restriction site selected Kinetics of induction different Try a longer time course for induction than than expected the 4 5 hours recommended Not induced at OD oy 0 4 0 6 Induce expression at OD soo 0 4 0 6 IPTG solution is too old Prepare a fresh solution of IPTG or use up to 10 mM IPTG Protein is difficult to detect on Perform a western blot using the a Coomassie stained gel Anti Xpress antibody for detection Purification Introduction ProBond Binding Capacity of ProBond Scale up of Expression for Purification on ProBond Once you have expressed your recombinant fusion protein you may purify your fusion protein using a metal chelating resin such as ProBond available from Invitrogen Cat no R801 01 ProBond is a nickel charged Sepharose resin that can be used for affinity purification of fusion proteins containing the 6xHis tag Proteins bound to the resin may be eluted with either low pH buffer or competition with imidazole or histidine e To scale up your pilot expression for purification see below e To purify your fusion protein using ProBond refer to the ProBond Purification System manual for instruction The ProBond Purification System manual is availabl
13. body to your protein of interest In addition the Positope Control Protein Cat no R900 50 is available from Invitrogen for use as a positive control for detection of fusion proteins containing an Xpress or HisG epitope The ready to use WesternBreeze Chromogenic Kits and WesternBreeze Chemiluminescent Kits are available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods For more information please refer to our website www invitrogen com or call Technical Support see page 18 1 Inoculate 2 ml of SOB containing ampicillin 50 pg ml and chloramphenicol 35 pg ml with a single recombinant E coli colony Grow overnight at 37 C with shaking 225 rpm 2 The next day inoculate 25 ml of SOB to an OD q of 0 1 with the overnight culture Antibiotics are not required for expression Please note that you may increase the volume to produce more protein 3 Grow the culture at 37 C with shaking 225 rpm to an OD soo 0 40 6 4 Add IPTG to a final concentration of 1 mM 0 25 ml of 100 mM IPTG stock to 25 ml culture 5 Grow the culture at 37 C with vigorous shaking for the optimal time determined in pilot expression see page 9 6 Harvest the cells by centrifugation and either proceed directly to lysis or freeze the cells at 80 C until ready for use Use the information provided in the table below to troubleshoot your expression Expression experiment Problem Pro
14. claving 5 After autoclaving add antibiotic if desired Add chloramphenicol to a final concentration of 10 ug ml and ampicillin to a final concentration of 50 pg ml Continued on next page Recipes continued Antibiotics 100 mM IPTG 50 mM CaCl Ampicillin Prepare a stock solution of 50 mg ml in deionized water and filter sterilize it with a 0 22 pm filter To prepare selective medium cool medium to 50 C after autoclaving and add 1 ml of the ampicillin stock per liter of media both liquid and solid for a final concentration of 50 ug ml Store the stock solution at 20 C Chloramphenicol Prepare a stock solution of 35 mg ml in 100 ethanol It is not necessary to filter sterilize Store the stock solution at 20 C To prepare selective medium cool the medium to 50 C after autoclaving and add 1 ml of the stock solution per liter of medium for a final concentration of 35 pg ml For 10 ml of a 100 mM solution Dissolve 0 24 g of IPTG in sterile deionized water Bring the final volume to 10 m and filter sterilize 0 22 um filter Do not autoclave For 100 ml of a50 mM solution Dissolve 0 56 g of anhydrous CaCl MW 111 in 100 ml of deionized water Filter sterilize 0 22 um filter or autoclave Use this solution ice cold for competent cell preparation 13 Map of pRSET A B and C pRSET A B and C The map below shows the features of pRSET A B and C The complete sequence of t
15. domain and the recombinant protein allows for subsequent removal of this N terminal fusion peptide from the purified recombinant protein Expression of the gene of interest from pRSET is controlled by the strong phage T7 promoter that drives expression of gene 10 10 T7 RNA polymerase specifically recognizes this promoter For expression of the gene of interest it is necessary to deliver T7 RNA polymerase to the cells by either inducing expression of the polymerase using the gratuitous inducer isopropyl B D thiogalactoside IPTG or infecting the cell with phage expressing the polymerase Once sufficient T7 RNA polymerase is produced it binds to the T7 promoter and transcribes the gene of interest The BL21 DE3 pLysS strain is specifically included in the kit for expression of T7 regulated genes This strain carries the DE3 bacteriophage lambda lysogen This lambda lysogen contains the lacl gene the T7 RNA polymerase gene under control of the lacUV5 promoter and a small portion of the lacZ gene This lac construct is inserted into the int gene which inactivates the int gene Disruption of the int gene prevents excision of the phage i e lysis in the absence of helper phage The lac repressor represses expression of T7 RNA polymerase Addition of IPTG allows expression of T7 RNA polymerase The BL21 DE3 pLysE strain is also available For more information on this strain BL21 DE3 and BL21 DE3 pLysS see page 3 Continued on
16. e for downloading at www invitrogen com To purify your fusion protein using another metal chelating resin refer to the manufacturer s instructions TM One milliliter of ProBond binds at least 1 mg of recombinant protein This amount can vary depending on the nature of the protein TM Please note that the capacity of ProBond is about 1 mg of protein per milliliter Depending on the expression level of your recombinant fusion protein you may need to adjust the culture volume to bind the maximum amount of recombinant fusion protein to your column For a prepacked 2 ml ProBond column start with 50 ml of bacterial culture If you need to purify larger amounts of recombinant protein you may need more ProBond resin See page v for ordering information To grow and induce a 50 ml bacterial culture 1 Inoculate 10 ml of SOB or LB containing 50 100 pg ml ampicillin and 34 ug ml chloramphenicol if needed with a single recombinant E coli colony Grow overnight at 37 C with shaking 225 250 rpm to OD 1 2 The next day inoculate 50 ml of SOB or LB containing 50 100 pg ml ampicillin with 1 ml of the overnight culture Note You can scale up further and inoculate all of the 10 ml overnight culture into 500 ml of medium but TM you may need a larger bed volume for your ProBond column 4 Grow the culture at 37 C with shaking 225 250 rpm to an OD soo 0 5 2 3 hours The cells should be in mid log phase
17. ene 10 sequence Provides protein stability N terminal Xpress epitope tag Allows detection of the fusion protein by the Xpress Antibody Cat no R910 25 or the Xpress HRP Antibody Cat no R911 25 Enterokinase cleavage site Provides a site for efficient removal of the fusion tag Multiple cloning site Allows insertion of your gene of interest and facilitates in cloning in frame with the N terminal epitope tag T7 reverse priming site Allows sequencing of the insert T7 terminator Permits efficient transcription termination fl origin Allows single strand rescue of DNA bla promoter Allows expression of the ampicillin resistance gene Ampicillin resistance gene B lactamase Allows selection of the plasmid in E coli pUC origin High copy replication and growth in E coli 15 Map of pRSET lacZ Description PRSET lacZ is a 5911 bp control vector expressing B galactosidase Note that galactosidase is fused to an N terminal peptide containing the Xpress peptide 6xHis tag and an enterokinase recognition site The molecular weight is approximately 120 kDa The figure below summarizes the features of the PRSET lacZ vector The complete sequence of the vector is available for downloading from our website at www invitrogen com or from Technical Support see page 18 RBS ATG 6xHis Xpress Epitope EK lacz stop Comments for pRSET lac
18. he vector is available for downloading from our website at www invitrogen com or from Technical Support see page 18 Comments for pRSET A 2897 nucleotides Version C does not contain Sac T7 promoter bases 20 39 6xHis tag bases 112 129 T7 gene 10 leader bases 133 162 Xpress epitope bases 169 192 Multiple cloning site bases 202 248 T7 reverse priming site bases 295 314 T7 transcription terminator bases 256 385 f1 origin bases 456 911 bla promoter bases 943 1047 Ampicillin bla resistance gene ORF bases 1042 1902 pUC origin bases 2047 2720 C Features of pRSET A B and C Features The important elements of pRSET A B and C are described in the table below All features have been functionally tested Feature Benefit T7 promoter Provides tight dose dependent regulation of heterologous gene expression Provides a binding site for most T7 promoter primers for sequencing into the insert Ribosome binding site Optimally spaced from the multiple cloning site for efficient translation of the gene of interest Initiation ATG Provides a translational initiation site for the fusion protein N terminal 6xHis tag Permits purification of recombinant fusion protein on metal chelating resins Le ProBond In addition it allows detection of the recombinant protein with the Anti HisG Antibody R940 25 or the Anti HisG HRP Antibody Cat no R941 25 T7 g
19. her expressed or implied including any warranty of merchantability or fitness for a particular purpose 18 Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 30 T7 Expression System This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany The composition and or use of this product may be claimed in U S Patent No 5 693 489 licensed to Life Technologies Corporation by Brookhaven Science Associates LLC The T7 expression system is based on technology developed at Brookhaven National Laboratory under contract with the U S Department of Energy and is the subject of patents and patent applications assigned to Brookhaven Science Associates LLC BSA By provisions of the Distribution License Agreement granted to Life Technologies covering said patents and patent applications Life Technologies grants you a non exclusive sub license under patents assigned to BSA for the use of this technology including the enclosed materials based upon the following conditions 1 these materials are to be used for non commercial research purposes only A separate license u
20. ification For native and denaturing purification 6 purifications K850 01 System of recombinant proteins Continued on next page Accessory Products Electrophoresis Products Media and Reagents vi A large variety of pre cast polyacrylamide gels and electrophoresis products are available separately from Invitrogen for the separation and analysis of recombinant proteins Ordering information for the most widely used products is provided below For more detailed information including size concentration and well formats available for pre cast gel systems visit www invitrogen com or contact Technical Support page 18 Product Quantity Cat no NuPAGE Novex 4 12 Bis Tris Gels 1 box 10 gels NP0321BOX Novex 10 Tris Glycine Gels 1 box 10 gels EC6075BOX NuPAGE LDS Sample Buffer 4X 10 ml NP0007 250 ml NP0008 Novex Tris Glycine SDS Sample Buffer 2X 20 ml LC2676 SimplyBlue Safe Stain 1L LC6060 Colloidal Blue Staining Kit 1 kit LC6025 XCell SureLock Mini Cell amp XCell II Blot 1 unit EI0002 Module In addition to the pre cast polyacrylamide gel systems Invitrogen offers a wide range of pre mixed media and reagents Ordering information for the most widely used products is provided below For more detailed information visit www invitrogen com or contact Technical Support page 18 Product Quantity Cat no S 0 C Medium
21. invitrogen by technologies pRSET A B and C For high level expression of recombinant proteins in E coli Cat no V351 20 Rev Date 18 June 2010 Manual part no 25 0213 MAN00000061 ii Table of Contents Kit Contents and Storage EEN iv Accessory ProduetS enee dieses eene edn kann de eeen an rennende v Introduction zessen gern gaen eeen eee 1 Kee REENEN EE 1 MethodS canrenseeinererngennneeenenniddeneen EE 3 General Come seess Eeer 3 Cloning into pRSET A B and CERN 4 opreegt tee ee ebe Eeer 8 Purification EE 11 el DE 12 EE 12 Map of pRSET A B and Cen 14 Features of pRSET A B and C EE 15 Ma p ot pRSET Aleedung Eed ees 16 Transformation Protocol for TOP10F and BL21 DE3 pLysS EE 17 Technical Support siteni ieioea tten ehesek nsa ESAN anani deene Ereegnes 18 P rchaser Notification enin e eee a a e aaa a a ae e RRR E 19 iii Kit Contents and Storage Kit Contents Shipping and Storage Long Term Storage This kit contains the following reagents 20 ug each of pRSET A B and C in TE buffer pH 8 0 40 ul each at 0 5 ug ul 1 stab TOP10F 1 stab BL21 DE3 pLysS 1 stab BL21 DE3 pLysS containing the pRSET lacZ control TE buffer pH 8 0 10 mM Tris HCl 1 mM EDTA pH 8 0 This kit is shipped on wet ice Upon receipt store the plasmids at 20 C and the stabs at 4 C For long term storage of E coli strains supplied as stabs with this kit prepare glycerol stocks as follows 1
22. ion 8 Purify your recombinant protein by chromatography on metal 11 chelating resin e g ProBond Methods General Cloning Introduction E coli Host Maintaining pRSETA B and C The following information is provided to help you clone your gene of interest into pRSET A B and C For basic information on DNA ligations E coli transformations restriction analysis DNA sequencing and DNA biochemistry see Current Protocols in Molecular Biology Ausubel et al 1994 For cloning and transformation we recommend using a recA end A strain such as TOP10F included in the kit TOP10F cells are recA and endA making them suitable for cloning propagation and maintenance Genotype of TOP10F F lacl4 Tn10 Tet mcrA mrr hsdRMS mcrBC 80lacZ M15 lac 74 recA1 araD139 ara leu 7697 galU galK rpsL Str endA1 nupG BL21 DE3 pLysS is specifically designed for expression of genes regulated by the T7 promoter Do not use this strain for propagation or maintenance of your plasmid Genotype of BL21 DE3 pLysS E ompT hsdSB ry my gal dem DE3 pLysS Cam To propagate and maintain pRSET A B and C use the supplied 0 5 ug ul stock solution in TE pH 8 0 to transform a recA endA E coli strain like TOP10F DH5a T1 8 TOP10 or equivalent Select transformants on LB plates containing 50 100 pg ml ampicillin Be sure to prepare a glycerol stock of a transformant containing plasmid for
23. logies Corporation or their respective owners 20 invitrogen by Lefe technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
24. long term storage see page 7 Cloning into pRSET A B and C Introduction The multiple cloning site of each version of pRSET is provided below and on the following pages see pages 5 6 To generate recombinant proteins that are expressed correctly and contain the N terminal fusion peptide it is necessary to clone in frame with the N terminal peptide To facilitate cloning the pRSET vector is provided in three different reading frames They differ only in the spacing between the sequences that code for the N terminal peptide and the multiple cloning site For proper expression determine which restriction sites are appropriate for ligation Multiple Cloning Below is the multiple cloning site for pRSET A Restriction sites are labeled to Site of pRSET A indicate the actual cleavage site The boxed nucleotides indicate the variable 21 91 148 205 262 region Sequencing and functional testing have confirmed the multiple cloning site The complete sequence of pRSET A is available for downloading at www invitrogen com or from Technical Support see page 18 For a map and description of the features of pRSET A please refer to pages 14 15 T7 promoter RBS l AATACGACTC ACTATAGGGA GACCACAACG GTTTCCCTCT AGAAATAATT TTGTTTAACT TTAAGAAGGA Polyhistidine 6xHis region l 1 GATATACAT ATG CGG GGT TCT CAT CAT CAT CAT CAT CAT GGT ATG GCT AGC ATG ACT Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr
25. ml of SOB containing ampicillin 50 ug ml and chloramphenicol 35 pg ml with a single recombinant E coli colony Grow overnight at 37 C with shaking The next day inoculate 25 ml of SOB it is not necessary to include antibiotics for expression to an OD sop of 0 1 with the overnight culture Grow the culture at 37 C with vigorous shaking to an Oo 0 4 0 6 Remove a 1 ml aliquot of cells prior to IPTG induction centrifuge the sample in a microcentrifuge and aspirate the supernatant Freeze the cell pellet at 20 C This will be the time zero sample Add IPTG to a final concentration of 1 mM 0 25 ml of 100 mM IPTG stock to 25 ml culture and continue to grow the cells See page 12 for preparation of the IPTG stock solution After 1 hour of incubation remove a 1 ml sample centrifuge as described in Step 4 aspirate the supernatant and freeze the cell pellet at 20 C Continue to take samples at 1 hour intervals for 4 to 6 hours When all time points have been collected resuspend each pellet in 100 pl of 20 mM phosphate buffer at neutral pH and freeze in liquid nitrogen or methanol dry ice exercise caution when handling liquid nitrogen it can cause severe burns if it comes in contact with the skin wear appropriate protective equipment Thaw the frozen lysate at 42 C Repeat this freeze thaw two to three additional times and pellet the insoluble protein in a microcentrifuge for 10 minutes at maximum speed at 4 C Remove the
26. nder patents owned by BSA is required for any commercial use including the use of these materials for research purposes or production purposes by any commercial entity Information about commercial license may be obtained from The Office of Technology Transfer Brookhaven National Laboratory Bldg 475D P O Box 5000 Upton New York 11973 5000 Phone 516 344 7134 2 No materials that contain the cloned copy of the T7 gene 1 the gene for T7 RNA polymerase may be distributed further to third parties outside of your laboratory unless the recipient receives a copy of this sub license and agrees to be bound by its terms This limitation applies to strains BL21 DE3 BL21 DE3 pLysS and BL21 DE3 pLysE CE6 BL21 SI Competent Cells and any derivatives that are made of them You may refuse this sub license by returning this product unused in which case Life Technologies accept return of the product with a full refund By keeping or using this product you agree to be bound by the terms of this license 19 Reference Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Techno
27. on LB containing 35 pg ml chloramphenicol and 50 pg ml ampicillin Chloramphenicol selects for maintenance of the pLysS plasmid required for T7 lysozyme expression and ampicillin selects for the pRSET plasmid see Appendix for media recipes It is important to maintain BL21 DE3 pLysS strains on LB and chloramphenicol as loss of the plasmid will increase basal levels of transcription We recommend preparing a frozen glycerol stock of untransformed BL21 DE3 pLysS see page 7 Plasmid DNA may be prepared using your method of choice We recommend the S N A P MiniPrep Kit Cat no K1900 01 or the PureLink HiPure Plasmid DNA Purification Kit Cat no K2100 01 for isolation of pure plasmid DNA Included in the kit is a stab of E coli strain BL21 DE3 pLysS containing PRSET lacZ pRSET lacZ is pRSET A with the B galactosidase gene cloned into the BamH I and Hind III sites for use as a positive control for expression B galactosidase should appear as a band of approximately 120 kDa on a denaturing polyacrylamide gel The complete sequence of this vector is available at www invitrogen com or from Technical Support page 18 Continued on next page Expression continued Pilot Expression Analysis of Samples Note Expression conditions will vary depending on the nature of your protein therefore we recommend performing a time course experiment to optimize expression of your recombinant protein 1 10 Inoculate 2
28. rvices Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whet
29. that you sequence your construct to confirm that your gene is in frame with the N terminal tag and in the proper orientation The T7 promoter primer Cat no N560 02 is available for sequencing your insert in pRSET A B or C 1 Grow 1 2 ml of the E coli strain to be frozen in SOB medium overnight with antibiotic selection when appropriate 2 Combine 0 85 ml of the overnight culture with 0 15 ml of sterile glycerol sterilized by autoclaving Mix well by vortexing Transfer to an appropriate freezing vial preferably a screw cap air tight gasket 5 Freeze in an ethanol dry ice bath or liquid nitrogen and then transfer to 80 C for long term storage Expression Introduction Preparation for Expression Plasmid Preparation Positive Control Vector BL21 DE3 pLysS cells are included with the kit as the host for expression You will need pure plasmid DNA of your construct to transform into BL21 DE3 pLysS for expression studies Since each recombinant protein has different characteristics that may affect optimal expression it is helpful to do a pilot expression to determine the best conditions for optimal expression of your particular protein To express your recombinant protein from pRSET transform the plasmid into BL21 DE3 pLysS and select for ampicillin resistant transformants see page 17 Before proceeding with the expression streak out the BL21 DE3 pLysS transformant containing the recombinant plasmid
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